Granulocyte-colony stimulating factor receptors

ABSTRACT

Mammalian granulocyte-colony stimulating factor (G-CSF) receptor proteins, DNAs and expression vectors encoding mammalian G-CSF receptors, and processes for producing mammalian G-CSF receptors as products of recombinant cell culture, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. Ser. No. 08/006,183, filed Jan.15, 1993, now U.S. Pat. No. 5,422,248, which is a continuation of U.S.Ser. No. 07/587,329, filed Sep. 24, 1990, now abandoned, which is acontinuation-in-part of U.S. application Ser. No. 522,952, filed Apr. 3,1990, now abandoned which is a continuation-in-part of U.S. applicationSer. No. 416,306, now abandoned filed Oct. 3, 1989, which is acontinuation-in-part of U.S. application Ser. No. 412,816, filed on Sep.26, 1989 now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates generally to cytokine receptors and morespecifically to granulocyte-colony stimulating factor receptors.

Human Granulocyte-Colony Stimulating Factor (G-CSF) is alineage-specific hematopoietic protein which stimulates theproliferation and differentiation of granulocyte-committed progenitorcells. Human G-CSF has also been shown to functionally activate matureneutrophils. The cDNAs for human (Nagata et al., Nature 319;415, 1986)and mouse G-CSF (Tsuchiya et al., PNAS 83, 7633, 1986) have beenisolated, permitting further structural and biological characterizationof G-CSF.

G-CSF initiates its biological effect on cells by binding to specificG-CSF receptor protein expressed on the plasma membrane of a G-CSFresponsive cell. Because of the ability of G-CSF to specifically bindG-CSF receptor (G-CSFR), purified G-CSFR compositions will be useful indiagnostic assays for G-CSF, as well as in raising antibodies to G-CSFreceptor for use in diagnosis and therapy. In addition, purified G-CSFreceptor compositions may be used directly in therapy to bind orscavenge G-CSF, thereby providing a means for regulating the immuneactivities of this cytokine. In order to study the structural andbiological characteristics of G-CSFR and the role played by G-CSFR inthe responses of various cell populations to G-CSF or other cytokinestimulation, or to use G-CSFR effectively in therapy, diagnosis, orassay, purified compositions of G-CSFR are needed. Such compositions,however, are obtainable in practical yields only by cloning andexpressing genes encoding the receptors using recombinant DNAtechnology. Efforts to purify the G-CSFR molecule for use in biochemicalanalysis or to clone and express mammalian genes encoding G-CSFR havebeen impeded by lack of a suitable source of receptor protein or mRNA.Prior to the present invention, no cell lines were known to express highlevels of G-CSFR constitutively and continuously, which precludedpurification of receptor for sequencing or construction of geneticlibraries for direct expression cloning.

SUMMARY OF THE INVENTION

The present invention provides DNA sequences encoding mammaliangranulocyte-colony stimulating factor receptors (G-CSFR) or subunitsthereof. Preferably, such DNA sequences are selected from the groupconsisting of (a) cDNA clones having a nucleotide sequence derived fromthe coding region of a native G-CSFR gene; (b) DNA sequences which arecapable of hybridization to the cDNA clones of (a) under moderatelystringent conditions and which encode biologically active G-CSFRmolecules; and (c) DNA sequences which are degenerate as a result of thegenetic code to the DNA sequences defined in (a) and (b) and whichencode biologically active G-CSFR molecules. The present invention alsoprovides recombinant expression vectors comprising the DNA sequencesdefined above, recombinant G-CSFR molecules produced using therecombinant expression vectors, and processes for producing therecombinant G-CSFR molecules using the expression vectors.

The present invention also provides isolated or purified proteincompositions comprising mammalian G-CSFR. Preferred G-CSFR proteins aresoluble forms of the native receptors.

The present invention also provides compositions for use in therapy,diagnosis, assay of G-CSFR, or in raising antibodies to G-CSFR,comprising effective quantities of soluble native or recombinantreceptor proteins prepared according to the foregoing processes. Theseand other aspects of the present invention will become evident uponreference to the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows restrictions maps of cDNA clones D-7 and 25-1 containingregions encoding human G-CSFR proteins.

FIGS. 2A-C depicts that cDNA sequence of clone D-7 which was isolatedfrom a human placental library, and the predicted amino acid sequence ofthis clone. The coding region of the predicted mature full-lengthmembrane-bound protein from clone D7 is defined by amino acids 1-759.The predicted N-terminal Glu of the mature protein is designated aminoacid number 1 and is underlined. The putative transmembrane region atamino acids 604-629 is also underlined.

FIG. 3 depicts the 3' nucleotide sequence and predicted C-terminal aminoacid sequence of clone 25-1, which is the result of an alternativesplicing arrangement. The position of the intron insertion in clone 25-1is indicated at with a ▾ after nucleotide 2411 of FIG. 1. The positionof the intron-exon boundaries are indicated with a ▾, and splice-donorand splice-acceptor recognition sequences are boxed. Sequences alsopresent in clone D-7 are underlined.

DETAILED DESCRIPTION OF THE INVENTION Definitions

G-CSF is a growth factor which induces growth and differentiation ofneutrophilic granulocyte progenitors. The biological activities of G-CSFare mediated through binding to specific cell surface receptors,referred to as "G-CSF receptors" or "G-CSFR". G-CSFR, as used herein,refers to proteins having amino acid sequences which are substantiallysimilar to native mammalian G-CSFR amino acid sequences, such as thehuman G-CSFR sequence disclosed in FIG. 1, or fragments thereof, andwhich are biologically active as defined below, in that they are capableof binding G-CSF molecules or, in their native configuration as intacthuman plasma membrane proteins, transducing a biological signalinitiated by a G-CSF molecule binding to a cell, or cross-reacting withanti-G-CSFR antibodies raised against G-CSFR from natural (i.e.,nonrecombinant) sources. Specific embodiments of G-CSFR includepolypeptides substantially equivalent to the sequence of amino acids1-759 of FIG. 2 (clone D-7) or the sequence of amino acids 1-776 of theprotein encoded by clone 25-1 as disclosed in FIGS. 2 and 3. The terms"G-CSF receptor" or "G-CSFR" include, but are not limited to, solubleG-CSF receptors, as defined below. As used throughout thisspecification, the term "mature" means a protein expressed in a formlacking a leader sequence as may be present in full-length transcriptsof a native gene. Various bioequivalent protein and amino acid analogsare described in detail below.

The mature N-terminal amino acid is predicted to be Glu¹ (underlined anddesignated as amino acid 1 in FIG. 2), based on the algorithm of vonHeijne, G., Nucl. Acids Res. 14:4683 (1986), for determining signalcleavage sites. However, several factors suggest that Ser³ may be thecorrect mature N-terminal amino acid, based on the observation that Ser³is 21 amino acids from the N-terminal Met and is preceded by the smallamino acid residue Gly, both of which are accepted criteria foridentifying signal cleavage sites. The actual N-terminal amino acid ofthe mature protein can be confirmed by sequencing purified G-CSFRprotein using standard techniques. Thus, amino acid sequences equivalentto those described above include, for example, amino acids -3 through759 of FIG. 2 (clone D-7) or -3 through 776 of the protein encoded byclone 25-1 as disclosed in FIGS. 2 and 3.

In their native configuration, receptor proteins are present as intacthuman plasma membrane proteins having an extracellular region whichbinds to a ligand, a hydrophobic transmembrane region which causes theprotein to be immobilized within the plasma membrane lipic bilayer, anda cytoplasmic or intracellular region which interacts with cytoplasmicproteins and/or chemicals to deliver a biological signal to effectorcells via a cascade of chemical reactions within the cytoplasm of thecell. The hydrophobic transmembrane region and a highly charged sequenceof amino acids in the cytoplasmic region immediately following thetransmembrane region cooperatively function to hilt transport of theG-CSFR across the plasma membrane. "Soluble G-CSFR" or "sG-CSFR", asused in the context of the present invention, refer to a protein, or asubstantially equivalent analog, having an amino acid sequencecorresponding to the extracellular region of native G-CSFR, for examplepolypeptides having the amino acid sequences substantially equivalent tothe sequences of amino acids 1-603 of FIG. 2. Equivalent sG-CSFRsinclude polypeptides which vary from the sequences shown in FIG. 2 byone or more substitutions, deletions, or additions, and which retain theability to bind G-CSF and inhibit the ability of G-CSF to transduce asignal via cell surface bound G-CSF receptor proteins. Because sG-CSFRproteins are devoid of a transmembrane region, they are secreted fromthe host cell in which they are produced. Equivalent soluble G-CSFRinclude, for example, the sequence of amino acids -3 through 603 of FIG.2. When administered in therapeutic formulations, sG-CSFR proteinscirculate in the body and bind to circulating G-CSF molecules,preventing interaction of G-CSF with natural G-CSF receptors andinhibiting transduction of G-CSF-mediated biological signals, such asimmune or inflammatory responses. The ability of a polypeptide toinhibit G-CSF signal transduction can be determined by transfectingcells with recombinant G-CSF receptor DNAs to obtain recombinantreceptor expression. The cells are then contacted with G-CSF and theresulting metabolic effects examined. If an effect results which isattributable to the action of the ligand, then the recombinant receptorhas signal transducing activity. Examplary procedures for determiningwhether a polypeptide has signal transducing activity are disclosed byIdzerda et al., J. Exp. Med. 171:861 (1990); Curtis et al., Proc. Natl.Acad. Sci. USA 86:3045 (1989); Prywes et al., EMBO J. 5:2179 (1986); andChou et al., J. Biol. Chem. 262:1842 (1987). Alternatively, primarycells of cell lines which express an endogenous G-CSF receptor and havea detectable biological response to G-CSF could also be utilized.

"Substantially similar" G-CSFR include those whose amino acid or nucleicacid sequences vary from a reference sequence by one or moresubstitutions, deletions, or additions, the net effect of which is toretain biological activity of the G-CSFR protein. Alternatively, nucleicacid subunits and analogs are "substantially similar" to the specificDNA sequences disclosed herein if: (a) the DNA sequence is derived fromthe coding region of a native mammalian G-CSFR gene; (b) the DNAsequence is capable of hybridization to DNA sequences of (a) undermoderately stringent conditions and which encode biologically activeG-CSFR molecules; or DNA sequences which are degenerate as a result ofthe genetic code to the DNA sequences defined in (a) or (b) and whichencode biologically active G-CSFR molecules. Substantially similaranalog proteins will be greater than about 30 percent similar to thecorresponding sequence of the native G-CSFR. Sequences having lesserdegrees of similarity but comparable biological activity are consideredto be equivalents. More preferably, the analog proteins will be greaterthan about 80 percent similar to the corresponding sequence of thenative G-CSFR, in which case they are defined as being "substantiallyidentical." In defining nucleic acid sequences, all subject nucleic acidsequences capable of encoding substantially similar amino acid sequencesare considered substantially similar to a reference nucleic acidsequence. Percent similarity may be determined, for example, bycomparing sequence information using the GAP computer program, version6.0, available from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman(Adv. Appl. Math. 2:482, 1981). Briefly, the GAP program definessimilarity as the number of aligned symbols (i.e., nucleotides or aminoacids) which are similar, divided by the total number of symbols in theshorter of the two sequences. The preferred default parameters for theGAP program include: (1) a unary comparison matrix (containing a valueof 1 for identities and 0 for non-identifies) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, ed., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

"Recombinant," as used herein, means that a protein is derived fromrecombinant (e.g., microbial or mammalian) expression systems."Microbial" refers to recombinant proteins made in bacterial or fungal(e.g., yeast) expression systems. As a product, "recombinant microbial"defines a protein produced in a microbial expression system which isessentially free of native endogenous substances. Protein expressed inmost bacterial cultures, e.g., E. coli, will be free of glycan. Proteinexpressed in yeast may have a glycosylation pattern different from thatexpressed in mammalian cells.

"Biologically active," as used throughout the specification as acharacteristic of G-CSF receptors, means that a particular moleculeshares sufficient amino acid sequence similarity with the embodiments ofthe present invention disclosed herein to be capable of bindingdetectable quantities of G-CSF, transmitting a G-CSF stimulus to a cell,for example, as a component of a hybrid receptor construct, orcross-reacting with anti-G-CSFR antibodies raised against G-CSFR fromnatural (i.e., nonrecombinant) sources. Preferably, biologically activeG-CSF receptors within the scope of the present invention are capable ofbinding greater than 0.1 nmoles G-CSF per nmole receptor, and mostpreferably, greater than 0.5 nmole G-CSF per nmole receptor in standardbinding assays (see below).

"DNA sequence" refers to a DNA polymer, in the form of a separatefragment or as a component of a larger DNA construct, which has beenderived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesequence and its component nucleotide sequences by standard biochemicalmethods, for example, using a cloning vector. Such sequences arepreferably provided in the form of an open reading frame uninterruptedby internal nontranslated sequences, or introns, which are typicallypresent in eukaryotic genes. Genomic DNA containing the relevantsequences could also be used. Sequences of non-translated DNA may bepresent 5' or 3' from the open reading frame, where the same do notinterfere with manipulation or expression of the coding regions.

"Nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides.DNA sequences encoding the proteins provided by this invention can beassembled from cDNA fragments and short oligonucleotide linkers, or froma series of oligonucleotides, to provide a synthetic gene which iscapable of being expressed in a recombinant transcriptional unit.

"Recombinant expression vector" refers to a replicable DNA constructused either to amplify or to express DNA which encodes G-CSFR and whichincludes a transcriptional unit comprising an assembly of (1) a geneticelement or elements having a regulatory role in gene expression, forexample, promoters or enhancers, (2) a structural or coding sequencewhich is transcribed into mRNA and translated into protein, and (3)appropriate transcription and translation initiation and terminationsequences. Structural elements intended for use in yeast expressionsystems preferably include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, whererecombinant protein is expressed without a leader or transport sequence,it may include an N-terminal methionine residue. This residue mayoptionally be subsequently cleaved from the expressed recombinantprotein to provide a final product.

"Recombinant microbial expression system" means a substantiallyhomogeneous monoculture of suitable host microorganisms, for example,bacteria such as E. coli or yeast such as S. cerevisiae, which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit as a component of aresident plasmid. Generally, cells constituting the system are theprogeny of a single ancestral transformant. Recombinant expressionsystems as defined herein will express heterologous protein uponinduction of the regulatory elements linked to the DNA sequence orsynthetic gene to be expressed.

The term "isolated", as used in the context of this specification todefine the purity of a G-CSFR or sG-CSFR protein or protein composition,means that the protein or protein composition is substantially free ofother proteins of natural or endogenous origin and contains less thanabout 1% by mass of protein contaminants residual of productionprocesses. Such compositions, however, can contain other proteins addedas stabilizers, carriers, excipients or co-therapeutics. G-CSFR orsG-CSFR is isolated if it is detectable as a single protein band in apolyacrylamide gel by silver staining.

Isolation of cDNAs Encoding G-CSFR

The coding sequence of a mammalian G-CSFR is obtained by first isolatinga cDNA sequence encoding G-CSFR from a recombinant DNA library generatedusing either genomic DNA or cDNA. The preferred method for constructinga cDNA library is to prepare polyadenylated mRNA obtained from aparticular cell line which expresses a mammalian G-CSFR and convertingthe polyadenylated RNA to cDNA by reverse transcription. A particularlypreferred cellular source of mRNA for construction of the cDNA libraryis human placental RNA.

A cDNA library will contain G-CSFR sequences which can be readilyidentified by screening the library with an appropriate nucleic acidprobe which is capable of hybridizing with G-CSFR cDNA. Such probes canbe derived from the nucleotide sequences disclosed herein.Alternatively, DNAs encoding G-CSFR proteins can also be assembled byligation of synthetic oligonucleotide subunits to provide a completecoding sequence.

The cDNAs encoding G-CSFR of the present invention were isolated by themethod of direct expression cloning. Specifically, a cDNA library wasconstructed by first isolating cytoplasmic mRNA from human placentaltissue using standard techniques. Polyadenylated mRNA was isolated andused to prepare double-stranded cDNA. Purified cDNA fragments were thenligated into psfCAV vector DNA described in detail below in Example 2.The psfCAV vectors containing the G-CSFR cDNA fragments were transformedinto E. coli strain DH5a. Transformants were plated to provideapproximately 800 colonies per plate. The resulting colonies wereharvested and each pool used to prepare plasmid DNA for transfectioninto COS-7 cells essentially as described by Cosman et al. (Nature312:768, 1984) and Luthman et al. (Nucl. Acid Res. 11:1295, 1983).Transformants expressing biologically active cell surface G-CSFreceptors were identified by screening for the ability of G-CSFR to bind¹²⁵ I-G-CSF (5×10⁻¹⁰ M). Specifically, transfected COS-7 cells wereincubated with medium containing ¹²⁵ I-G-CSF, the cells washed to removeunbound labeled G-CSF, and the cell monolayers contacted with X-ray filmto detect concentrations of G-CSF binding, as disclosed by Sims et al,Science 241:585 (1988). Transfectants detected in this manner appear asdark foci against a relatively light background.

This approach as used to screen approximately 30,000 cDNAs in pools ofapproximately 600 cDNAs until assay of a transfectant pool indicatedpositive foci for G-CSF binding. A frozen stock of bacteria from thispositive pool was grown in culture and plated to provide individualcolonies, which were screened until single clones were identified whichare capable of directing synthesis of a surface protein with detectableG-CSF binding activity. Additional cDNA clones can be isolated from cDNAlibraries of other mammalian species by cross-species hybridization ofhuman G-CSFR cDNAs with cDNA derived from other mammalian species. Foruse in hybridization, DNA encoding G-CSFR may be covalently labeled witha detectable substance such as a fluorescent group, a radioactive atomor a chemiluminescent group by methods well known to those skilled inthe art. Such probes could also be used for in vitro diagnosis ofparticular conditions.

Like most mammalian genes, mammalian G-CSF receptors are presumablyencoded by multi-exon genes. Alternative mRNA constructs which can beattributed to different mRNA splicing events following transcription,and which share large regions of identity or similarity with the cDNAsclaimed herein, are considered to be within the scope of the presentinvention.

Proteins and Analogs

The present invention provides isolated recombinant mammalian G-CSFRpolypeptides as defined above. Isolated G-CSFR polypeptides aresubstantially free of other contaminating materials of natural orendogenous origin and contain less than about 1% by mass of proteincontaminants residual of production processes. Such polypeptides areoptionally without associated native-pattern glycosylation. MammalianG-CSFR of the present invention includes, by way of example, primate,human, murine, canine, feline, bovine, ovine, equine and porcine G-CSFR.Derivatives of G-CSFR within the scope of the invention also includevarious structural forms of the primary protein which retain biologicalactivity. Due to the presence of ionizable amino and carboxyl groups,for example, a G-CSFR protein may be in the form of acidic or basicsalts, or may be in neutral form. Individual amino acid residues mayalso be modified by oxidation or reduction.

The primary amino acid structure may be modified by forming covalent oraggregative conjugates with other chemical moieties, such as glycosylgroups, lipids, phosphate, acetyl groups and the like, or by creatingamino acid sequence mutants. Covalent derivatives are prepared bylinking particular functional groups to G-CSFR amino acid side chains orat the N- or C-termini. Other derivatives of G-CSFR within the scope ofthis invention include covalent or aggregative conjugates of G-CSFR orits fragments with other proteins or polypeptides, such as by synthesisin recombinant culture as N-terminal or C-terminal fusions. For example,the conjugated peptide may be a a signal (or leader) polypeptidesequence at the N-terminal region of the protein whichco-translationally or post-translationally directs transfer of theprotein from its site of synthesis to its site of function inside oroutside of the cell membrane or wall (e.g., the yeast α-factor leader).G-CSFR protein fusions can comprise peptides added to facilitatepurification or identification of G-CSFR (e.g., poly-His). The aminoacid sequence of G-CSF receptor can also be linked to the peptideAsp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (Hopp et al., Bio/Technology6:1204,1988.) The latter sequence is highly antigenic and provides anepitope reversibly bound by a specific monoclonal antibody, enablingrapid assay and facile purification of expressed recombinant protein.This sequence is also specifically cleaved by bovine mucosalenterokinase at the residue immediately following the Asp-Lys pairing.Fusion proteins capped with this peptide may also be resistant tointracellular degradation in E. coli.

G-CSFR derivatives may also be used as immunogens, reagents inreceptor-based immunoassays, or as binding agents for affinitypurification procedures of G-CSF or other binding ligands. G-CSFRderivatives may also be obtained by cross-linking agents, such asM-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, atcysteine and lysine residues. G-CSFR proteins may also be covalentlybound through reactive side groups to various insoluble substrates, suchas cyanogen bromide-activated, bisoxirane-activated,carbonyldiimidazole-activated or tosyl-activated agarose structures, orby adsorbing to polyolefin surfaces (with or without glutaraldehydecross-linking). Once bound to a substrate, G-CSFR may be used toselectively bind (for purposes of assay or purification) anti-G-CSFRantibodies or G-CSF.

The present invention also includes G-CSFR with or without associatednative-pattern glycosylation. G-CSFR expressed in yeast or mammalianexpression systems, e.g., COS-7 cells, may be similar or slightlydifferent in molecular weight and glycosylation pattern than the nativemolecules, depending upon the expression system. Expression of G-CSFRDNAs in bacteria such as E. coli provides non-glycosylated molecules.Functional mutant analogs of mammalian G-CSFR having inactivatedN-glycosylation sites can be produced by oligonucleotide synthesis andligation or by site-specific mutagenesis techniques. These analogproteins can be produced in a homogeneous, reduced-carbohydrate form ingood yield using yeast expression systems. N-glycosylation sites ineukaryotic proteins are characterized by the amino acid triplet Asn-A₁-Z, where A₁ is any amino acid except Pro, and Z is Ser or Thr. In thissequence, asparagine provides a side chain amino group for covalentattachment of carbohydrate. Such a site can be eliminated bysubstituting another amino acid for Asn or for residue Z, deleting Asnor Z, or inserting a non-Z amino acid between A₁ and Z, or an amino acidother than Asn between Asn and A₁.

G-CSFR derivatives may also be obtained by mutations of G-CSFR or itssubunits. A G-CSFR mutant, as referred to herein, is a polypeptidehomologous to G-CSFR but which has an amino acid sequence different fromnative G-CSFR because of a deletion, insertion or substitution.

Bioequivalent analogs of G-CSFR proteins may be constructed by, forexample, making various substitutions of residues or sequences ordeleting terminal or internal residues or sequences not needed forbiological activity. For example, aliphatic amino acid residues, such asIle, Val, Leu or Ala may be substituted for one another, or polar aminoacid residues, such as Lys and Arg, Glu and Asp, or Gln and Asn, may besubstituted for one another. Also, cysteine residues can be deleted orreplaced with other amino acids to prevent formation of incorrectintramolecular disulfide bridges upon renaturation. Other approaches tomutagenesis involve modification of adjacent dibasic amino acid residuesto enhance expression in yeast systems in which KEX2 protease activityis present. Generally, substitutions should be made conservatively;i.e., the most preferred substitute amino acids are those havingphysicochemical characteristics resembling those of the residue to bereplaced. Similarly, when a deletion or insertion strategy is adopted,the potential effect of the deletion or insertion on biological activityshould be considered.

Subunits of G-CSFR may be constructed by deleting terminal or internalresidues or sequences. Particularly preferred subunits include those inwhich the transmembrane region and intracellular domain of G-CSFR aredeleted or substituted with hydrophilic residues to facilitate secretionof the receptor into the cell culture medium. The resulting protein is asoluble truncated G-CSFR molecule which may retain its ability to bindG-CSF.

Mutations in nucleotide sequences constructed for expression of analogG-CSFR must, of course, preserve the reading frame phase of the codingsequences and preferably will not create complementary regions thatcould hybridize to produce secondary mRNA structures such as loops orhairpins which would adversely affect translation of the receptor mRNA.Although a mutation site may be predetermined, it is not necessary thatthe nature of the mutation per se be predetermined. For example, inorder to select for optimum characteristics of mutants at a given site,random mutagenesis may be conducted at the target codon and theexpressed G-CSFR mutants screened for the desired activity.

Not all mutations in the nucleotide sequence which encodes G-CSFR willbe expressed in the final product, for example, nucleotide substitutionsmay be made to enhance expression, primarily to avoid secondarystructure loops in the transcribed mRNA (see EPA 75,444A, incorporatedherein by reference), or to provide codons that are more readilytranslated by the selected host, e.g., the well-known E. coli preferencecodons for E. coli expression.

Mutations can be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981); andU.S. Pat. Nos. 4,518,584 and 4,737,462 disclose suitable techniques, andare incorporated by reference herein.

Expression of Recombinant G-CSFR

The present invention provides recombinant expression vectors whichinclude synthetic or cDNA-derived DNA fragments encoding mammalianG-CSFR or bioequivalent analogs operably linked to suitabletranscriptional or translational regulatory elements derived frommammalian, microbial, viral or insect genes. Such regulatory elementsinclude a transcriptional promoter, an optional operator sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences which control the termination oftranscription and translation, as described in detail below. The abilityto replicate in a host, usually conferred by an origin of replication,and a selection gene to facilitate recognition of transformants mayadditionally be incorporated. DNA regions are operably linked when theyare functionally related to each other. For example, DNA for a signalpeptide (secretory leader) is operably linked to DNA for a polypeptideif it is expressed as a precursor which participates in the secretion ofthe polypeptide; a promoter is operably linked to a coding sequence ifit controls the transcription of the sequence; or a ribosome bindingsite is operably linked to a coding sequence if it is positioned so asto permit translation. Generally, operably linked means contiguous and,in the case of secretory leaders, contiguous and in reading frame.

DNA sequences encoding mammalian G-CSF receptors which are to beexpressed in a microorganism will preferably contain no introns thatcould prematurely terminate transcription of DNA into mRNA; however,premature termination of transcription may be desirable, for example,where it would result in mutants having advantageous C-terminaltruncations, for example, deletion of a transmembrane region to yield asoluble receptor not bound to the cell membrane. Due to code degeneracy,there can be considerable variation in nucleotide sequences encoding thesame amino acid sequence. Other embodiments include sequences capable ofhybridizing to the sequences of the provided cDNA under moderatelystringent conditions (50° C., 2 X SSC) and other sequences hybridizingor degenerate to those which encode biologically active G-CSF receptorpolypeptides.

Transformed host cells are cells which have been transformed ortransfected with G-CSFR vectors constructed using recombinant DNAtechniques. Transformed host cells ordinarily express G-CSFR, but hostcells transformed for purposes of cloning or amplifying G-CSFR DNA donot need to express G-CSFR. Expressed G-CSFR will be deposited in thecell membrane or secreted into the culture supernatant, depending on theG-CSFR DNA selected. Suitable host cells for expression of mammalianG-CSFR include prokaryotes, yeast or higher eukaryotic cells under thecontrol of appropriate promoters. Prokaryotes include gram negative orgram positive organisms, for example E. coli or bacilli. Highereukaryotic cells include established cell lines of mammalian origin asdescribed below. Cell-free translation systems could also be employed toproduce mammalian G-CSFR using RNAs derived from the DNA constructs ofthe present invention. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts aredescribed by Pouwels et al. (Cloning Vectors: A Laboratory Manual,Elsevier, New York, 1985), the relevant disclosure of which is herebyincorporated by reference.

Prokaryotic expression hosts may be used for expression of G-CSFR thatdo not require extensive proteolytic and disulfide processing.Prokaryotic expression vectors generally comprise one or more phenotypicselectable markers, for example a gene encoding proteins conferringantibiotic resistance or supplying an autotrophic requirement, and anorigin of replication recognized by the host to ensure amplificationwithin the host. Suitable prokaryotic hosts for transformation includeE. coli, Bacillus subtilis, Salmonella typhimurium, and various specieswithin the genera Pseudomonas, Streptomyces, and Staphyolococcus,although others may also be employed as a matter of choice.

Useful expression vectors for bacterial use can comprise a selectablemarker and bacterial origin of replication derived from commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017). Such commercial vectors include, forexample, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1(Promega Biotec, Madison, Wis., USA) and pCAV/NOT (ATCC Accession No.68014. These pBR322 "backbone" sections are combined with an appropriatepromoter and the structural sequence to be expressed. E. coli istypically transformed using derivatives of pBR322, a plasmid derivedfrom an E. coli species (Bolivar et al., Gene 2:95, 1977). pBR322contains genes for ampicillin and tetracycline resistance and thusprovides simple means for identifying transformed cells.

Promoters commonly used in recombinant microbial expression vectorsinclude the β-lactamase (penicillinase) and lactose promoter system(Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544,1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. AcidsRes. 8:4057, 1980; and EPA 36,776) and tac promoter (Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412,1982). A particularly useful bacterial expression system employs thephage λ P_(L) promoter and cI857ts thermolabile repressor. Plasmidvectors available from the American Type Culture Collection whichincorporate derivatives of the λ P_(L) promoter include plasmid pHUB2,resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E.coli RR1 (ATCC 53082).

Recombinant G-CSFR proteins may also be expressed in yeast hosts,preferably from the Saccharomyces species, such as S. cerevisiae. Yeastof other genera, such as Pichia or Kluyveromyces may also be employed.Yeast vectors will generally contain an origin of replication from the2μ yeast plasmid or an autonomously replicating sequence (ARS),promoter, DNA encoding G-CSFR, sequences for polyadenylation andtranscription termination and a selection gene. Preferably, yeastvectors will include an origin of replication and selectable markerpermitting transformation of both yeast and E. coli, e.g., theampicillin resistance gene of E. coli and S. cerevisiae trp1 gene, whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, and a promoter derived from a highlyexpressed yeast gene to induce transcription of a structural sequencedownstream. The presence of the trp1 lesion in the yeast host cellgenome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

Suitable promoter sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Suitable vectorsand promoters for use in yeast expression are further described in R.Hitzeman et al., EPA 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pBR322for selection and replication in E. coli (Amp^(r) gene and origin ofreplication) and yeast DNA sequences including a glucose-repressibleADH2 promoter and α-factor secretion leader. The ADH2 promoter has beendescribed by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier etal. (Nature 300:724, 1982). The yeast α-factor leader, which directssecretion of heterologous proteins, can be inserted between the promoterand the structural gene to be expressed. See, e.g., Kurjan et al., Cell30:933, 1982; and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330,1984. The leader sequence may be modified to contain, near its 3' end,one or more useful restriction sites to facilitate fusion of the leadersequence to foreign genes.

Suitable yeast transformation protocols are known to those of skill inthe art; an exemplary technique is described by Hinnen et al., Proc.Natl. Acad. Sci. USA 75:1929, 1978, selecting for Trp⁺ transformants ina selective medium consisting of 0.67% yeast nitrogen base, 0.5%casamino acids, 2% glucose, 10 μg/ml adeninc and 20 μg/ml uracil.

Host strains transformed by vectors comprising the ADH2 promoter may begrown for expression in a rich medium consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs upon exhaustion ofmedium glucose. Crude yeast supernatants are harvested by filtration andheld at 4° C. prior to further purification.

Various mammalian or insect cell culture systems can be employed toexpress recombinant protein. Baculovirus systems for production ofheterologous proteins in insect cells are reviewed by Luckow andSummers, Bio/Technology 6:47 (1988). Examples of suitable mammalian hostcell lines include the COS-7 lines of monkey kidney cells, described byGluzman (Cell 23:175, 1981), and other cell lines capable of expressingan appropriate vector including, for example, L cells, C127, 3T3,Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalianexpression vectors may comprise nontranscribed elements such as anorigin of replication, a suitable promoter and enhancer linked to thegene to be expressed, and other 5' or 3' flanking nontranscribedsequences, and 5' or 3' nontranslated sequences, such as necessaryribosome binding sites, a polyadenylation site, splice donor andacceptor sites, and transcriptional termination sequences.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells may be provided byviral sources. For example, commonly used promoters and enhancers arederived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and humancytomegalovirus. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites may be used to provide the other genetic elementsrequired for expression of a heterologous DNA sequence. The early andlate promoters are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 vital originof replication (Fiers et al., Nature 273:113, 1978). Smaller or largerSV40 fragments may also be used, provided the approximately 250 bpsequence extending from the Hind III site toward the BglI site locatedin the viral origin of replication is included. Further, mammaliangenomic G-CSFR promoter, control and/or signal sequences may beutilized, provided such control sequences are compatible with the hostcell chosen. Additional details regarding the use of a mammalian highexpression vector to produce a recombinant mammalian G-CSF receptor areprovided in Example 2 below. Exemplary vectors can be constructed asdisclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983).

A useful system for stable high level expression of mammalian receptorcDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986).

A particularly preferred eukaryotic vector for expression of G-CSFR DNAis disclosed below in Example 2. This vector, referred to as pCAV/NOT,was derived from the mammalian high expression vector pDC201 andcontains regulatory sequences from SV40, adenovirus-2, and humancytomegalovirus.

Purified mammalian G-CSF receptors or analogs are prepared by culturingsuitable host/vector systems to express the recombinant translationproducts of the DNAs of the present invention, which are then purifiedfrom culture media or cell extracts.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.For example, a suitable affinity matrix can comprise a G-CSF or lectinor antibody molecule bound to a suitable support. Alternatively, ananion exchange resin can be employed, for example, a matrix or substratehaving pendant diethylaminoethyl (DEAE) groups. The matrices can beacrylamide, agarose, dextran, cellulose or other types commonly employedin protein purification. Alternatively, a cation exchange step can beemployed. Suitable cation exchangers include various insoluble matricescomprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups arepreferred.

Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a G-CSFR composition. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture is usually isolated byinitial extraction from cell pellets, followed by one or moreconcentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. Finally, high performance liquid chromatography(HPLC) can be employed for final purification steps. Microbial cellsemployed in expression of recombinant mammalian G-CSFR can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents.

Fermentation of yeast which express mammalian G-CSFR as a secretedprotein greatly simplifies purification. Secreted recombinant proteinresulting from a large-scale fermentation can be purified by methodsanalogous to those disclosed by Urdal et al. (J. Chromatog. 296:171,1984). This reference describes two sequential, reversed-phase HPLCsteps for purification of recombinant human GM-CSF on a preparative HPLCcolumn.

Human G-CSFR synthesized in recombinant culture is characterized by thepresence of non-human cell components, including proteins, in amountsand of a character which depend upon the purification steps taken torecover human G-CSFR from the culture. These components ordinarily willbe of yeast, prokaryotic or non-human higher eukaryotic origin andpreferably are present in innocuous contaminant quantities, on the orderof less than about 1 percent by weight. Further, recombinant cellculture enables the production of G-CSFR free of proteins which may benormally associated with G-CSFR as it is found in nature in its speciesof origin, e.g. in cells, cell exudates or body fluids.

G-CSFR compositions are prepared for administration by mixing G-CSFRhaving the desired degree of purity with physiologically acceptablecarriers. Such carriers will be nontoxic to recipients at the dosagesand concentrations employed. Ordinarily, the preparation of suchcompositions entails combining the G-CSFR with buffers, antioxidantssuch as ascorbic acid, low molecular weight (less than about 10residues) polypeptides, proteins, amino acids, carbohydrates includingglucose, sucrose or dextrins, chelating agents such as EDTA, glutathioneand other stabilizers and excipients.

G-CSFR compositions may be used to attenuate G-CSF-mediated immuneresponses. To achieve this result, a therapeutically effective quantityof a G-CSFR composition is administered to a mammal, preferably a human,in association with a pharmaceutical carrier or diluent.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1

Binding Assays

A. Radiolabeling of G-CSF. Recombinant human G-CSF, in the form of afusion protein containing a hydrophilic octapeptide at the N-terminus,was expressed in yeast as a secreted protein and purified by affinitychromatography as described by Hopp et al., Bio/Technology 6:1204, 1988.The protein was radiolabeled using the commercially available solidphase agent, IODO-GEN (Pierce). In this procedure, 5 μg of IODO-GEN wereplated at the bottom of a 10×75 mm glass tube and incubated for 20minutes at 4° C. with 75 μl of 0.1M sodium phosphate, pH 7.4 and 20 μl(2 mCi) Na ¹²⁵ I. This solution was then transferred to a second glasstube containing 5 μg G-CSF in 45 μl PBS for 20 minutes at 4° C. Thereaction mixture was fractionated by gel filtration on a 2 ml bed volumeof Sephadex G-25 (Sigma) equilibrated in Roswell Park Memorial Institute(RPMI) 1640 medium containing 2.5% (w/v) bovine serum albumin (BSA),0.2% (w/v) sodium azide and 20 mM Hepes pH 7.4 (binding medium). Thefinal pool of ¹²⁵ I-G-CSF was diluted to a working stock solution of1×10⁻⁷ M in binding medium and stored for up to one month at 4° C.without detectable loss of receptor binding activity. The specificactivity is routinely 1×10¹⁶ cpm/mmole G-CSF. Radiolabeled G-CSF is usedas described below to assay for G-CSF receptors.

B. Membrane Binding Assays. Human placental membranes were incubated at4° C. for 2 hr with ¹²⁵ I-G-CSF in binding medium, 0.1% bacitracin,0.02% aprotinin, and 0.4% BSA in a total volume of 1.2 ml. Control tubescontaining in addition a 100×molar excess of unlabeled G-CSF were alsoincluded to determine non-specific binding. The reaction mixture wasthen centrifuged at 15,000x g in a microfuge for 5 minutes. Supernatantswere discarded, the surface of the membrane pellets carefully rinsedwith ice-cold binding medium, and the radioactivity counted on a gammacounter. Using this assay, it was determined that the G-CSFR present inthe COS cell supernatants of Example 2 had a K_(a) of about 1×10⁹ M⁻¹and a molecular weight of about 35 kDa.

C. Solid Phase Binding Assays. The ability of G-CSFR to be stablyadsorbed to nitrocellulose from detergent extracts of human cells yetretain G-CSF-binding activity provided a means of detecting G-CSFR.Cells extracts were prepared by mixing a cell pellet with a 2X volume ofPBS containing 1% Triton X-100 and a cocktail of protease inhibitors (2mM phenylmethyl sulfonyl fluoride, 10 μM pepstatin, 10 μM leupeptin, 2mM o-phenanthroline and 2 mM EGTA) by vigorous vortexing. The mixturewas incubated on ice for 30 minutes after which it was centrifuged at12,000x g for 15 minutes at 8° C. to remove nuclei and other debris. Twomicroliter aliquots of cell extracts were placed on dry BA85/21nitrocellulose membranes (Schleicher and Schuell, Keene, N.H.) andallowed to dry. The membranes were incubated in tissue culture dishesfor 30 minutes in Tris (0.05M) buffered saline (0.15M) pH 7.5 containing3% w/v BSA to block nonspecific binding sites. The membrane was thencovered with 0.3 nM ¹²⁵ I-G-CSF in PBS+3% BSA and incubated for 2 hr at4° C. with shaking. At the end of this time, the membranes were washed 3times in PBS, dried and placed on Kodak X-Omat AR film for 18 hr at -70°C. This assay was performed to detect the presence of G-CSFR in variouscells lines and tissue sources.

D. Binding Assay for Soluble G-CSFR. Soluble G-CSFR present in COS-7cell supernatants are measured by inhibition of ¹²⁵ I-CSF binding to aG-CSF-dependent cell line, or any other human cell or cell lineexpressing G-CSF receptors, such as as human placental cell.Supernatants are harvested from COS-7 cells 3 days after transfection,concentrated 10-fold, and preincubated with ¹²⁵ I-G-CSF for 1 hour at37° C. Appropriate G-CSF-receptor-bearing cells are added to a finalvolume of 150 ul, incubated for an additional 30 minutes at 37° C., andassayed and analyzed as described by Park et al., J. Biol. Chem.261:4177 (1986).

Example 2

Isolation of Human G-CSF R cDNAs by Direct Expression of Active Proteinin COS-7 Cells

A tissue source for G-CSFR was selected by screening various human celllines and tissues for expression of G-CSFR based on their ability tobind ¹²⁵ I-labeled G-CSF, prepared as described above in Example 1A.Human placental membranes were found to express a reasonable number ofreceptors. Equilibrium binding studies were performed according toExample 1B and showed that the membrane exhibited biphasic binding of¹²⁵ I-G-CSF with high affinity sites (K_(a) =4×10¹⁹ M⁻¹) of 0.4 pmolesreceptor/mg protein.

An unsized cDNA library was constructed by reverse transcription ofpolyadenylated mRNA isolated from total RNA extracted from the humanplacental tissue (Ausubel et al., eds., Current Protocols in MolecularBiology, Vol. 1, 1987). The cells were harvested by lysing the tissuecells in a guanidinium isothiocyanate solution and total RNA wasisolated using standard techniques as described by Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982.

Polyadenylated RNA was isolated by oligo dT cellulose chromatography anddouble-stranded cDNA was prepared by a method similar to that of Gublerand Hoffman, Gene 25:263, 1983. Briefly, the polyadenylated RNA wasconvened to an RNA-cDNA hybrid with reverse transcriptase using oligo dTas a primer. The RNA-cDNA hybrid was then converted into double-strandedcDNA using RNAase H in combination with DNA polymerase I. The resultingdouble stranded cDNA was blunt-ended with T4 DNA polymerase. BglIIadaptors were ligated to the 5' ends of the resulting blunt-ended cDNAas described by Haymerle, et al., Nuclear Acids Research, 14:8615, 1986.The non-ligated adaptors were removed by gel filtration chromatographyat 68° C., leaving 24 nucleotide non-self-complementary overhangs on thecDNA. The same procedure was used to convert the 5' BglII ends of themammalian expression vector psfCAV to 24 nucleotide overhangscomplementary to those added to the cDNA. Optimal proportions ofadaptored vector and cDNA were ligated in the presence of T4polynucleotide kinase. Dialyzed ligation mixtures were electroporatedinto E. coli strain DH5α and transformants selected on ampicillinplates.

The resulting cDNAs were ligated into the eukaryotic expression vectorpsfCAV, which was designed to express cDNA sequences inserted at itsmultiple cloning site when transfected into mammalian cells. psfCAV wasassembled from pDC201 (a derivative of pMLSV, previously described byCosman et al., Nature 312: 768, 1984), SV40 and cytomegalovirus DNA andcomprises, in sequence with the direction of transcription from theorigin of replication: (1) SV40 sequences from coordinates 5171-5270containing the origin of replication, enhancer sequences and early andlate promoters; (2) cytomegalovirus sequences containing the promoterand enhancer regions (nucleotides 671 to +63 from the sequence publishedby Boechan et al. (Cell 41:521, 1985); (3) adenovirus-2 sequences fromcoordinates 5779-6079 containing sequences for the motor late promoterand the first exon of the tripartite leader (TPL), coordinates 7101-7172and 9634-9693 containing the second exon and part of the third exon ofthe TPL and a multiple cloning site (MCS) containing sites for XhoI,KpnI, SmaI and BglI; (4) SV40 sequences from coordinates 4127-4100 and2770-2533 containing the polyadenylation and termination signals forearly transcription; (5) with adenovirus sequences from coordinates10532-11156 of the virus-associated RNA genes VAI and VAII of pDC201;and (6) pBR322 sequences from coordinates 4363-2486 and 1094-375containing the ampicillin resistance gene and origin of replication.

The resulting human placental cDNA library in sfCAV was used totransform E. coli strain DH5α, and recombinants were plated to provideapproximately 500-600 colonies per plate and sufficient plates toprovide approximately 30,000 total colonies per screen. Colonies werescraped from each plate, pooled, and plasmid DNA prepared from eachpool. The pooled DNA was then used to transfect a sub-confluent layer ofmonkey COS-7 cells using DEAE-dextran followed by chloroquine treatment,as described by Luthman et al., Nucl. Acids Res. 11:1295 (1983) andMcCutchan et al., J. Natl. Cancer Inst. 41:351 (1986). The cells werethen grown in culture for three days to permit transient expression ofthe inserted sequences. After three days, cell culture supernatants werediscarded and the cell monolayers in each plate assayed for G-CSFbinding as follows. Three ml of binding medium containing 1.2×10⁻¹¹ M¹²⁵ I-labeled flag-G-CSF was added to each plate and the platesincubated at 4° C. for 120 minutes. This medium was then discarded, andeach plate was washed once with cold binding medium (containing nolabeled G-CSF) and twice with cold PBS. The edges of each plate werethen broken off, leaving a flat disk which was contacted with X-ray filmfor 72 hours at -70° C. using an intensifying screen. G-CSF bindingactivity was visualized on the exposed films as a dark spot against arelatively uniform background.

After approximately 30,000 recombinants from the library had beenscreened in this manner, nine transfectant pools were observed toprovide G-CSF binding foci which were clearly apparent against thebackground exposure.

A frozen stock of bacteria from the positive pool was then used toobtain plates of approximately 60 colonies. Replicas of these plateswere made on nitrocellulose filters, and the plates were then scrapedand plasmid DNA prepared and transfected as described above to identifya positive plate. Bacteria from individual colonies from thenitrocellulose replica of this plate were grown in 0.2 ml cultures,which were used to obtain plasmid DNA. The plasmid DNA was thentransfected into COS-7 cells as described above. In this manner, asingle clone, clone D-7, was isolated which was capable of inducingexpression of G-CSFR in COS cells. A glycerol stock of bacteriatransformed with this G-CSFR cDNA clone in the expression vectorpCAV/NOT (or pDC302) has been deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852, USA, underaccession number 68102 (deposited Sep. 27, 1989).

An additional cDNA clone encoding G-CSFR was isolated from the sameplacental library. Recombinants from the placental cDNA library wereplated on E. coli strain DH5α and transformants selected on ampicillinplates. The transformants were screened by plaque hybridizationtechniques under conditions of high stringency (63° C., 0.2X SSC) usinga ³² P-labeled probe made from the human G-CSFR clone D-7. A hybridizingclone (clone 25-1) was isolated which is identical to clone D-7, exceptthat it contains an intron insertion after nucleotide 2411, addingnucleotides 2412-2832 of FIG. 3 and resulting in a change in readingframe and a corresponding change in amino acid sequence. The 3'nucleotide sequence and predicted C-terminal amino acid sequence ofclone 25-1 are set forth in FIG. 3.

Example 3

Construction of cDNAs Encoding Soluble Human G-CSFR

Soluble human G-CSFR was cloned into the mammalian expression vectorpDC302, described above, utilizing the polymerase chain reaction (PCR)technique. The following primers were used:

5' End Primer

5'-GGTACCATGGCAAGGCTGGGAAAC

Asp718 site/Initiation Codon

3' End Primer

5'-TCTAGAACTCAGCCTCGATGTG

BglII/Termination Codon

The PCR product thus contains Asp718 and BglII restriction sites at the5' and 3' termini, respectively. These restriction sites are used toclone into pDC302. The 3' sequence is antisense relative to sequencedisclosed in FIG. 2. The template for the PCR reaction is clone 25-1,described above, which contains the G-CSFR. The DNA sequences encodingthe G-CSFR are then amplified by PCR, substantially as described byInnis et al., eds., PCR Protocols: A Guide to Methods and Applications(Academic Press, 1990). The resulting amplified clone was then isolatedand ligated into pDC302 and expressed in monkey COS-7 cells as describedabove.

Example 4

Preparation of Monoclonal Antibodies to G-CSFR

Preparations of purified recombinant G-CSFR, for example, human G-CSFR,or transfected COS cells expressing high levels of G-CSFR are employedto generate monoclonal antibodies against G-CSFR using conventionaltechniques, for example, those disclosed in U.S. Pat. No. 4,411,993.Such antibodies are likely to be useful in interfering with G-CSFbinding to G-CSF receptors, for example, in ameliorating toxic or otherundesired effects of G-CSF, or as components of diagnostic or researchassays for G-CSF or soluble G-CSF receptor.

To immunize mice, G-CSFR immunogen is emulsified in complete Freund'sadjuvant and injected in amounts ranging from 10-100 μg subcutaneouslyinto Balb/c mice. Ten to twelve days later, the immunized animals areboosted with additional immunogen emulsified in incomplete Freund'sadjuvant and periodically boosted thereafter on a weekly to biweeklyimmunization schedule. Serum samples are periodically taken byretro-orbital bleeding or tail-tip excision for testing by dot-blotassay (antibody sandwich) or ELISA (enzyme-linked immunosorbent assay).Other assay procedures are also suitable. Following detection of anappropriate antibody titer, positive animals are given an intravenousinjection of antigen in saline. Three to four days later, the animalsare sacrificed, splenocytes harvested, and fused to the murine myelomacell line NS1. Hybridoma cell lines generated by this procedure areplated in multiple microliter plates in a HAT selective medium(hypoxanthine, aminopterin, and thymidine) to inhibit proliferation ofnon-fused cells, myeloma hybrids, and spleen cell hybrids.

Hybridoma clones thus generated can be screened by ELISA for reactivitywith G-CSFR, for example, by adaptations of the techniques disclosed byEngvall et al., Immunochem. 8:871 (1971) and in U.S. Pat. No. 4,703,004.Positive clones are then injected into the peritoneal cavities ofsyngeneic Balb/c mice to produce ascites containing high concentrations(>1 mg/ml) of anti-G-CSFR monoclonal antibody. The resulting monoclonalantibody can be purified by ammonium sulfate precipitation followed bygel exclusion chromatography, and/or affinity chromatography based onbinding of antibody to Protein A of Staphylococcus aureus.

We claim:
 1. A purified biologically active Granulocyte ColonyStimulating Factor receptor (G-CSFR), selected from the group consistingof:(a) a G-CSFR having an amino acid sequence comprising amino acids 1through 759 of FIGS. 2A-2B; (b) a G-CSFR having an amino acid sequencecomprising amino acids 1 through 725 of FIGS. 2A-2B followed by aminoacids 726-776 of FIG. 3; (c) a biologically active G-CSFR identical tothe G-CSFR molecules of (a) or (b) except for modification(s) to theamino acid sequence selected from the group consisting of: inactivatedN-linked glycosylation sites; altered KEX2 protease cleavage sites; andconservative amino acid substitutions; and (d) soluble forms of theG-CSFR according to (a), (b) or (c), consisting of the extracellulardomain of G-CSFR,which G-CSFR molecules exhibit biological activity inan assay selected from the group consisting of a G-CSFR membrane bindingassay, a G-CSFR solid phase binding assay, a G-CSFR whole cell bindingassay, and a binding assay for soluble G-CSFR.
 2. A composition,comprising a G-CSFR according to claim 1, and a suitable diluent orcarrier.